The potential of plant biomass as a cheap and renewable substrate for the production of fuel and chemicals has gained considerable interest in recent years. The biological saccharification of cellulose, the main component of plant biomass, is of particular interest in the field of fuel ethanol production. At least four biologically mediated process steps are involved in the current cellulose-to-ethanol technology: (i) cellulose enzyme production; (ii) enzymatic saccharification of cellulose; (iii) fermentation of hexose sugars (end-products of cellulose hydrolysis); and (iv) fermentation of pentose sugars (end-products of hemicellulose hydrolysis) to ethanol. Lynd, L. R. et al., “Microbial cellulose utilization: fundamentals and biotechnology,” Microbiol. Mol. Biol. Rev. 66:506-577 (2002) Combining the four process steps above into a one-step conversion of cellulose to fuel ethanol (termed consolidated bioprocessing (CBP)) would result in a considerable reduction in processing costs See id.
The yeast Saccharomyces cerevisiae (S. cerevisiae) has superior ethanol formation properties, but is noncellulolytic. The expression of cellulases in S. cerevisiae would be a prerequisite for cellulose conversion via CBP. S. cerevisiae has received a great deal of interest regarding heterologous protein expression as well as the production of ethanol and other commodity products. See id.; Romanos, M. A. et al., “Foreign gene expression in yeast: a review,” Yeast 8:423-488 (1992) Expression of a functional cellulase system in S. cerevisiae would require the co-expression of at leak three groups of enzymes, namely endoglucanases (EC 3.2.1.4); exoglucanases (EC 3.2.1.91) and β-glucosidases (EC 3.2.1.21). These enzymes act synergistically to efficiently degrade cellulose Mansfield, S. D. and R. Meder, “Cellulose hydrolysis—the role of the mono-component cellulases in crystalline cellulose degradation,” Cellulose 10, 159-169 (2003)
Various cellulase genes have been expressed in S. cerevisiae with the aim of direct ethanol production from cellulose. Often, however, heterologous cellulase enzymes are produced by recombinant organisms in such low concentrations that the amount of saccharified substrate available is unable to sustain growth of the organisms. In an attempt to alleviate enzyme concentration deficiencies, yeast strains displaying cell surface proteins have been developed. Fujita, Y et al., “Direct and Efficient Production of Ethanol from Cellulosic Material with a Yeast Strain Displaying Cellulolytic Enzymes,” Applied and Environmental Microbiology 68: 5136-5141 (2002) describes an S. cerevisiae strain expressing tethered β-glucosidase I (BglI) and endoglucanase II (EgII). However, this strain, while able to grown on a linear, soluble polysaccharide, is unable to grown on insoluble celluose.
Improvements on such strains have been described and characterized, where the expression of four different tethered cellulase enzymes result in a strain having the capability of growth on insoluble cellulose. See, U.S. Application, entitled “Recombinant Yeast Strains Expressing Tethered Cellulase Enzyme,” to McBride et al., filed Nov. 20, 2007, and assigned to Dartmouth University, the entirety of which is herein incorporated by reference. In this previously-described strain, tethered versions of endoglucanase I (Eg1), cellobiohydrolase I (Cbh1), and cellobiohydrolase II (Cbh2) from Trichoderma reesei (T. reesei) and the β-glucosidase I (Bgl1) from Saccharomycopsis fibuligera (S. fibuligera) were used to transform S. cerevisiae. This tethered Eg1/Cbh1/Cbh2/Bgl1 transformed yeast strain was capable of growth on the insoluble cellulose substrate phosphoric acid swollen cellulose (PASC) and the crystalline insoluble cellulose substrate bacterial microcrystalline cellulose (BMCC).
Given that both the Eg1 and Cbh1 from the T. reesei have cellulose binding domains (CBDs) at their carboxy terminus, and that this is also where they are attached to the anchoring domain, these tethered constructs may not, however, necessarily provide sufficient activity on insoluble substrates. Additionally, T. reesei Cbh1 is typically not well secreted. While a codon optimized version may be somewhat improved, evidence suggests the improvement is not large if at all. Finally, tethered cellulase enzymes may not gain the access to the substrate that secreted versions do for stearic reasons. Thus, there is a need in the art to improve such tethered cellulase enzyme systems.
An additional approach to increase cellulose conversion via CBP in S. cerevisiae is to improve cellulose utilization by selection-based methods. Selection-based improvement of strains, including yeast strains, for improving cellulose utilization promises to be a powerful tool for engineering recombinant organisms for consolidated bioprocessing. However, to date, no demonstration of this technique has been accomplished.
Previous attempts to create strains built for selection experiments were not suitable for further experiments. This is due, in part, to the inability to separate the effect of amino acid utilization from cellulose utilization, and, in part, to the slow rate of growth rate of the previous strains which rendered them unsuitable for continuous culture because those strains were likely to wash out of the continuous culture at elevated dilution rates.
The solutions to these issues could come from a number of sources. First, prototrophic versions of these strains could be created, because these versions allow media to be formulated without adding any amino acids. When this is done, it can be calculated that the total carbon available to the cell in synthetic complete media (Yeast Nitrogen Base without amino acids from Difco) is 1.9 mg/L, all of which is present in vitamin components. This virtually eliminates concerns about the utilization of non-cellulose carbon sources during continuous cultures.
In addition to strain modification, an easier way to hydrolyze substrate other than Avicel PH105 is desired. However, such substrates are generally not available in large quantities, and producing them is prohibitively time consuming. Additionally, Avicel PH105 is easy to work with in well mixed systems and does not, for example, clog tubing. One other solution to the issue of slow growth rate and low cell concentration is to add soluble sugar as a co-feed in the system. This co-feed allows the cells to replicate at a relatively high rate and yet still to gain a selective benefit by being cellulolytic, since the soluble sugar concentration in the reactor can be kept very close to zero.
The present invention addresses the limitations of the systems described above. First, with regard to the improvement of tethered cellulase systems, the present invention provides for a transformed host cell with greater ability to grow on insoluble cellulose by the addition of a highly expressed, secreted Cbh1 to the Eg1/Cbh1/Cbh2/Bgl1 tethered system.
In addition, with regard to the selection-based approach, the present invention provides for a selection method and the creation of a new cellulolytic, prototrophic strain of S. cerevisiae utilizing this selection method. The new strain exhibits a number of phenotypic improvements with respect to cellulose utilization, including improved growth on mixes of Avicel and cellobiose, improved growth on bacterial microcrystalline cellulose (BMCC)-containing media, and biomass formation on solid Avicel containing media. Improved strains of the present invention attained cell counts on BMCC containing media about ten times faster than previously created strains.